Click Chemistry:  protocol for the labeling and modification of biomolecules

Click Chemistry is a reaction between azide and alkyne yielding covalent product - 1,5-disubstituted 1,2,3-triazole. This process is also known as CuAAC - Cu catalyzed alkyne azide cycloaddition.

Click Chemistry is based on copper catalysis. The catalyst is often introduced as Cu-TBTA complex.

Among the vast variety of organic reactions, Click Chemistry has been selected as a conjugation chemistry reaction because of several advantages.

• It is very selective. Click Chemistry reaction takes place only between azide and alkyne components. It is does not interfere with most any other organic groups present in DNA and proteins being labeled, such as amino and carboxy groups.

• There are no azides and alkynes in native biomolecules. These groups should be specially introduced into DNA and proteins. Alkyne-containing DNA can be prepared with alkyne phosphoramidite during standard oligo synthesis. Proteins labeled with azide and alkyne can be made using azide activated ester and alkyne activated ester.

• Click Chemistry takes place in water. Aqueous DMSO, DMF, acetonitrile, alcohols, or pure water and buffers can be used for the reaction. The reaction is biocompatible and can take place in living cells.

• Reaction is quick and quantitative. Click Chemistry is a tool that allows preparation of nanomols of conjugates in diluted solutions.

• The reaction is pH-insensitive. Unlike reaction of NHS esters with amines, and some other conjugation chemistries, there is no need to control pH in reaction mixture. There is no need to add any special buffer, acid or base - Click Chemistry works well in pH interval of 4-11.

• Protocol is simple! For example see our recommended DNA labeling protocol.

Click Chemistry thus became a tool for universal modification of DNA, proteins, conjugate preparation, and fluorescent labeling. Here are just a few examples.

• Fluorescent labeled oligonucleotides & dual-labeled probes for realtime PCR. We provide

alkyne phosphoramidites for easy synthesis of alkyne modified oligos, and fluorescent dye azides.

• Fluorescent & biotinylated DNA. Use alkynyl triphosphates for the incorporation of alkyne in DNA by PCR, termination, or nick translation. You can thereafter label DNA with any dye or biotin in your lab, without need in specific labeled triphosphates!

 • Fluorescent peptides, proteins, and antibodies. We provide alkyne and azide activated ester for the modification of proteins and peptides with either azide or alkyne. You can use alkyne- or azido-modified proteins for the preparation of conjugates with DNA, reporter groups and solid surfaces.

• Peptide-oligonucleotide conjugates. We provide azide activated ester for the labeling of peptides, and alkyne amidites for the synthesis of alkyno oligos.

• Biomolecules immobilized on nearly any solid phase. We would be glad to consult you on the modification of solid surfaces, provide you with custom solid phases, and guide you to success!

• Click Chemistry auxiliary reagents & catalysts are available in our catalogue.

• ... and nearly any other conjugates you can imagine can be done by Click Chemistry... Contact us to find how Click Chemistry can help you!

Some recent reviews on DNA modification with Click Chemistry:

A.V. Ustinov, I.A. Stepanova, V.V. Dubnyakova, T.S. Zatsepin, E.V. Nozhevnikova, V.A. Korshun. Modification of nucleic acids using [3+2]-dipolar cycloaddition of azides and alkynes. Russ. J. Bioorg. Chem., 36 (4), 401.445 (2010). http://www.springerlink.com/content/a623254qwg95107p/fulltext.pdf

A.H. El-Sagheer, T. Brown. Click chemistry with DNA. Chem. Soc. Rev., 39 (4), 1388.1405 (2010).

http://pubs.rsc.org/en/Content/ArticleLanding/2010/CS/b901971p#!divAbstract

F. Amblard, J.H. Cho, R.F. Schinazi. Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide, and oligonucleotide chemistry. Chem. Rev., 109 (9), 4207.4220 (2009).

http://pubs.acs.org/doi/pdf/10.1021/cr9001462

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